During its lifetime, a cell spends a considerable fraction of its energy pumping sodium and calcium out and potassium in. This balancing process is similar to that found in the coils of the DNA double helix, where specific ions nestle and help stabilize this macromolecule. These are only two examples of selective ion interactions in biology; there are many others also vital to life. The existence of these interactions has been known since the early 20th century, when Franz Hofmeister observed that some salts (ionic compounds) aided the solution of proteins in egg, some caused proteins to destabilize and precipitate, and others ranged in activity between the two extremes. Hofmeister then ranked "salt-out" (destabilizing) ions versus "salt-in" (stabilizing) ions according to the magnitude of their effects (the "Hofmeister effects"). However, despite enormous effort, why certain interactions are preferred over others is not completely understood. Recently, a team of researchers from UC Berkeley used the model systems of acetate and formate (two simple carboxylic acids) with a series of cations to test predictions made in the literature for preferential interactions. Near-edge x-ray absorption fine structure (NEXAFS) spectroscopy was used as this technique is highly sensitive to the chemical environments around a molecule. Experiments at ALS Beamline 8.0.1 confirmed strengthening of the interaction between the cations and the carboxylate group in the following order: potassium, sodium, and lithium.

Cellular Preferences

We do not completely understand why the insides of our cells prefer potassium and our body uses valuable energy to pump sodium out, although they are very similar ions. To further explore this phenomenon, scientists performed experiments at ALS Beamline 8.0.1, where the local environment around models for ion-protein interactions were studied to see if new information could be gleaned from x-ray measurements. These experiments are uniquely sensitive to the local environment and interactions of the molecule of interest, but require intense monochromatic x-rays, such as provided by the ALS.

The team's measurements indicated carboxylate groups characteristic of proteins preferred sodium over potassium ions. These results lend strong experimental support to a critical part of the Law of Matching Water Affinities, a theory proposed by Kim Collins in 1997 that says that the least soluble pairs are formed by ions closest to each other in their hydration energy, or how strongly they hold on to water. Understanding the relative ranking of these ionic interactions (known as the Hofmeister series) will give researchers a deeper understanding of many biological and chemical processes, including protein unfolding and bubble coalescence.

A cluster used in a calculation of an acetate molecule interacting with sodium surrounded by waters. The spectra show a shift to higher in energy for sodium (blue), relative to potassium (red), indicative of a stronger interaction.

NEXAFS involves the promotion of a core-level electron to antibonding molecular orbitals and Rydberg states. Both of these are diffuse states that extend far beyond the molecule of interest, making them extremely sensitive to changes in molecular geometry and environment, such as the addition of nearby ions. The experiments were conducted in an aqueous solution in order to emulate biological conditions. NEXAFS is a high-vacuum technique, so several additional techniques were required to maintain low-enough pressures for experiments on the liquid samples, such as the liquid microjet, which was used to introduce the cations into the experiment. This key component permitted a vacuum state and provided a renewable sample, avoiding x-ray damage.

Using carbonyl (C=O) NEXAFS as a measuring stick, the group measured the selective interactions at the carbon K-edge in order to avoid the water signal that dominates oxygen K-edge spectra. For acetate, the carbon K-edge spectra for interactions with potassium, sodium, and lithium, respectively, were successively slightly higher in energy (blue shifted). For formate, there was a distinctly large shift between lithium and the other ions, whereas sodium and potassium were indistinguishable. The difference between acetate and formate was understood by the "Law of Matching Water Ion Affinities," a proposed explanation for Hofmeister effects that attributes the stability of ion pairs to their hydration pairing; that is, the difference between the hydration energies of acetate and formate relative to those of the alkali cations accounts for their dissimilarities in preferential interactions and thus the difference in spectral trends.

A density functional theory program (StoBe deMon) was used to calculate spectra as confirmation of the phenomenon. A molecular dynamics trajectory was run to gather information about the different positions the molecules could be in. The coordinates were then input into StoBe deMon, and spectra were calculated and averaged together to account for the different molecular motions on the spectra.

The team's results establish a way of probing selective interactions of ions with biological molecules in aqueous environments under relevant conditions and provide support for the Law of Matching Water Affinities, whßich invokes ion pairing to explain Hofmeister effects on proteins. Future work will involve additional testing of this phenomenon of selective interactions with +2 metals, and examining other ion-pairing interactions and other model systems such as amino acids, polypeptides, and DNA.